Interleukin-12 (IL-12), formerly known as cytotoxic lymphocyte maturation factor or natural killer cell stimulatory factor, is a 75-kDa (p75) heterodimeric cytokine composed of disulfide bonded 40-kDA (p40) and 35-kDa (p30) subunits. The p40 and p35 subunits are polypeptides which contain 306 amino acid residues and 197 amino acid residues, respectively (Gubler U., et al., Proc. Natl. Acad. Sci. USA, Vol. 88, 4143-4147 (1991)).
The p75 heterodimer is the biologically active form of IL-12 (Gubler, U., et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 4143; Wolf: S. F., et al., 1991, J. Immunol., 146: 3074). The IL-12 p75 heterodimer both activates and boosts cell mediated immune responses against foreign antigens by stimulating production of Th1 helper cells, stimulating activated T and natural killer (NK) cells, enhancing lytic activity of NK/LAK cells, and stimulating production of IFN-γ by resting and activated T and NK cells.
The p40 subunit of IL-12 has been shown to be produced in excess of the p35 subunit and is found in both monomeric and dimeric forms (Podlaski, F. J., et al., 1992, Arch. Biochem. Biophys. 294:230; D'Andrea, A., et al., 1992, J. Exp. Med., 176: 1387). IL-12 p40 homodimer is a potent IL-12 antagonist (Ling, P. et al., 1995, J. Immunol., 154: 116; Gillessen, S., et al., 1995, Eur. J. Immunol., 25: 200). In contrast to the p40 subunit, the p35 subunit of IL-12 has no known biological activity, and the p35 protein has only been found in association with the p40 subunit as part of the IL-12 p75 heterodimer. Therefore, there are two important types of epitopes presented by human IL-12: (1) epitopes presented by the p40 subunit; and (2) epitopes presented by three dimensional conformation of the IL-12 p75 heterodimer. Consequently, we designate antibodies that recognize epitopes present on the IL-12 p75 heterodimeric protein but do not recognize epitopes present on the IL-12 p40 subunit protein “heterodimer specific” antibodies.
It has been found that known IL-12 antibodies are not optimally effective in substantially neutralizing IL-12 bioactivity. IL-12 antibodies which immunologically react with the p40 subunit do not optimally block the bioactivity of human IL-12. For example, use of antibodies which react with epitopes presented by the p40 subunit is particularly problematic because production of IL-12 p75 heterodimer has been shown to result in excess inactive p40 subunits relative to bioactive p75 heterodimer (Podlaski, F. J., 1992, Arch. Biochem. Biophys. 294:230; D'Andrea, A. et al., 1992, J. Exp. Med., 176: 1387). As a result, the p40 antibodies are not as effective as heterodimer specific antibodies in reducing detrimental effects of IL-12 because the p40 subunit alone is not bioactive, and p40 antibodies tend to bind to the inactive p40 subunits rather than those p40 subunits that are part of a bioactive p75 heterodimer.
Even known antibodies which react only with the p75 heterodimer, do not effectively neutralize IL-12 bioactivity. For example, a previously identified IL-12 p75 heterodimer specific antibody, called 20C2 (Chizzonite et al., Cytokine, 6: A82a (1994) and D'Andrea et al., J. Exp. Med., Vol. 176, 1387-1398 (1992), cannot substantially block human IL-12 stimulated PHA-activated lymphoblast proliferation and IFN-γ production.
Heterodimer specific antibodies which more effectively neutralize IL-12 bioactivity are needed to reduce detrimental effects of IL-12. Increased levels of IL-12 in serum or tissue are known to be involved in the development and progression of autoimmune disorders. Thus, IL-12 antibodies are useful antagonists for controlling diseases with pathologies that are mediated through immune mechanisms, particularly, diseases associated with aberrant Th-1-type helper cell activity. Examples of such autoimmune disorders include multiple sclerosis, inflammatory bowel disease (IBD) including Crohn's disease and ulcerative colitis, rheumatoid arthritis and autoimmune diabetes mellitus. Other disease conditions which have been shown to benefit from the administration of IL-12 antibodies include transplantation/graft-versus-host disease septic shock.
In accordance with this invention, it has been found that IL-12 antibodies obtained from a mammal deficient in the gene encoding the p35 subunit and/or the gene encoding the p40 subunit substantially neutralize IL-12 bioactivity.